Abstract

Recent in silico studies suggested that the transcription cofactor LIM-only protein FHL2 is a major transcriptional regulator of mouse natural killer (NK) cells. However, the expression and role of FHL2 in NK cell biology are unknown. Here, we confirm that FHL2 is expressed in both mouse and human NK cells. Using FHL2-/- mice, we found that FHL2 controls NK cell development in the bone marrow and maturation in peripheral organs. To evaluate the importance of FHL2 in NK cell activation, FHL2-/- mice were infected with Streptococcus pneumoniae. FHL2-/- mice are highly susceptible to this infection. The activation of lung NK cells is altered in FHL2-/- mice, leading to decreased IFNγ production and a loss of control of bacterial burden. Collectively, our data reveal that FHL2 is a new transcription cofactor implicated in NK cell development and activation during pulmonary bacterial infection.

FHL2 expression in human and mouse natural killer (NK) cells. (A) Genome-wide expression analysis was performed on mouse cells using raw microarray data generated by the Immgen Consortium. The list of all Gene Expression Omnibus accession numbers and corresponding cell populations and series is available in Table S1 in Supplementary Material. (B–D,H) NK cells were purified from wild-type mouse spleens. (E–G) NK cells were purified from the peripheral blood of healthy donors. (B,E) FHL2 mRNA was analyzed using RT quantitative PCR and normalized to GAPDH mRNA in purified NK cells and in non-NK cells. The data are shown as the means ± SEM of at least three independent experiments. *p < 0.05 using the Mann–Whitney test. (C,F) Western blot analysis of NK cell lysates. Data are representative of three experiments. (H) NK cells were stimulated for 30 min with rmIL-15. (I) NK cells were purified from the peripheral blood of patients with a severe bacterial Community-Acquired Pneumonia. (D,G–I) FHL2 protein expression was assessed by immunofluorescence using an anti-FHL2 antibody, and DRAQ5™ was used to detect dsDNA. These panels show representative staining of at least two independent experiments.

Natural killer (NK) cell development in FHL2−/− mice. Flow cytometric analysis of NK cells in various organs of FHL2−/− and wild-type mice. (A,B) NK cells were defined as CD19− CD3ϵ− NKp46+ cells. (A) One representative experiment of the gating in different organs is shown. The percentage of NK cells in each organ is indicated. (B) Dots corresponding to the NK cell number and percentage for the indicated organs ± SEM of three experiments (n = at least 10 mice) are shown. (C) Flow cytometric analysis of NK1.1 and NKG2D expression and the CD11b+ percentage within CD19− CD3ϵ− NKp46+-gated spleen NK cells ± SEM of two distinct experiments are shown. (D) Gating strategy to identify the different stages of NK cell development in the bone marrow (BM). The percentages of cells in each of the specified gates are indicated. (E) Dots corresponding to the NK cell frequency for the indicated stage of development in the BM ± SEM of two distinct experiments are shown. (B,C,E) Each dot represents the data from one mouse. (B,C,E) *p < 0.05, **p < 0.01, ***p < 0.001 by Mann–Whitney test. (A–C) Data were confirmed using FHL2+/+ littermate mice.